Formulations of deoxycholic acid and salts thereof

ABSTRACT

The present application is directed to an aqueous pharmaceutical formulation comprising less than about 5% w/v sodium deoxycholate maintained at a pH sufficient to substantially inhibit precipitation of the sodium deoxycholate. Also disclosed herein, are methods for inhibiting precipitation of sodium deoxycholate in an aqueous solution comprising less than about 5% w/v of sodium deoxycholate, said method comprising maintaining pH of the solution of from at least about 8.0 to about 8.5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/716,070 which, inturns, claims the benefit under 35 U.S.C. §119(e) to U.S. provisionalapplication No. 61/274,129 which was converted from U.S. nonprovisionalapplication Ser. No. 12/397,229, filed Mar. 3, 2009, which isincorporated hereby by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations of acid inwater wherein the formulation is maintained at a pH such thatprecipitation of sodium deoxycholate is substantially inhibited.

BACKGROUND

Rapid removal of body fat is an age-old ideal, and many substances havebeen claimed to accomplish such results, although few have shownresults. “Mesotherapy,” or the use of injectables for the removal offat, is not widely accepted among medical practitioners due to safetyand efficacy concerns, although homeopathic and cosmetic claims havebeen made since the 1950's. Mesotherapy was originally conceived inEurope as a method of utilizing cutaneous injections containing amixture of compounds for the treatment of local medical and cosmeticconditions. Although mesotherapy was traditionally employed for painrelief, its cosmetic applications, particularly fat and celluliteremoval, have recently received attention in the United States. One suchreported treatment for localized fat reduction, which was popularized inBrazil and uses injections of phosphatidylcholine, has been erroneouslyconsidered synonymous with mesotherapy. Despite its attraction as apurported “fat-dissolving” injection, the safety and efficacy of thesecosmetic treatments remain ambiguous to most patients and physicians(see, Rotunda, A. M. and M. Kolodney, Dermatologic Surgery “Mesotherapyand Phosphatidylcholine Injections: Historical Clarification andReview”, 2006, 32: 465-480).

Recently published literature reports that the bile acid deoxycholicacid has fat removing properties when injected into fatty deposits invivo (See, WO 2005/117900 and WO 2005/112942, US2005/0261258;US2005/0267080; US2006/127468; and US2006/0154906). Deoxycholateinjected into fat tissue has the effects of: 1) degrading fat cells viaa cytolytic mechanism; and 2) causing skin tightening. Both of theseeffects are required to mediate the desired aesthetic corrections (i.e.,body contouring). The effects of deoxycholate into fat are spatiallycontained because deoxycholate injected into fat is rapidly inactivatedby exposure to protein, e.g. albumin, and then rapidly returns to theintestinal contents. As a result of this attenuation effect that confersclinical safety, fat removal therapies typically require 4-6 sessions.This localized fat removal without the need for surgery is beneficialnot only for therapeutic treatment relating to pathological localizedfat deposits (e.g., dyslipidemias incident to medical intervention inthe treatment of HIV), but also for cosmetic fat removal without theattendant risk inherent in surgery (e.g., liposuction) (see, Rotunda etal., Dermatol. Surgery “Detergent effects of sodium deoxycholate are amajor feature of an injectable phosphatidylcholine formulation used forlocalized fat dissolution”, 2004, 30: 1001-1008; and Rotunda et al., J.Am. Acad. Dermatol. “Lipomas treated with subcutaneous deoxycholateinjections”, 2005: 973-978).

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

SUMMARY

It has been found that aqueous solutions of sodium deoxycholate at low(i.e., <5% w/v) concentrations of sodium deoxycholate can be stabilizedby adjusting the pH of the solution. The present invention is directedto an aqueous pharmaceutical formulation comprising less than about 5%w/v sodium deoxycholate wherein the formulation is maintained at a pHsufficient to substantially inhibit precipitation of the sodiumdeoxycholate.

Also disclosed herein, are methods for inhibiting precipitation ofsodium deoxycholate in an aqueous solution comprising less than about 5%w/v of sodium deoxycholate, said method comprising maintaining pH of thesolution from about 8.0 to about 8.5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E and 1F show the variability in pH over a twoweek period of formulations containing 5, 50, 100 and 160 mg/mL (0.5, 5,10 and 16% w/v) sodium deoxycholate at 4° C. (FIGS. 1A, 1B and 1C) and25° C. (FIGS. 1D, 1E and 1F).

FIGS. 2A, 2B, 2C and 2D show that solubility is independent of lot tolot variability in pH. FIG. 2A is a comparison of the 5 mg (0.5% w/v)samples tested in FIGS. 1A, 1B, 1C, 1D, 1E and 1F. FIG. 2B is acomparison of the 50 mg (5% w/v) samples tested in FIGS. 1A, 1B, 1C, 1D,1E and 1F. FIG. 2C is a comparison of the 100 mg (10% w/v) samplestested in FIGS. 1A, 1B, 1C, 1D, 1E and 1F. FIG. 2D is a comparison ofthe 160 mg (16% w/v) samples tested in FIGS. 1A, 1B, 1C, 1D, 1E and 1F.

FIG. 3 shows a reverse phase HPLC chromatogram for a 20 mg/mL (2% w/v)aqueous formulation of sodium deoxycholate (0.9% NaCl and 0.9% benzylalcohol in 10 mM phosphate buffer at a pH of 8.0) after being incubatedfor 2 months at 4° C.

FIG. 4 shows the effect of temperature (4° C., 25° C. and 37° C.) on thepurity of various sodium deoxycholate formulations of over a two monthperiod.

FIG. 5 shows the effect of agitation on the purity of various sodiumdeoxycholate formulations of over a four hour period.

FIG. 6 shows the effect of UV exposure on the purity of various sodiumdeoxycholate formulations of over a 24 hour period.

FIG. 7 shows the effect of five freeze-thaw cycles on the purity ofvarious sodium deoxycholate formulations.

DETAILED DESCRIPTION

As used herein, certain terms have the following defined meanings

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.”. It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination when used for the intendedpurpose. Thus, a composition consisting essentially of the elements asdefined herein would not exclude trace contaminants from the isolationand purification method and pharmaceutically acceptable carriers, suchas phosphate buffered saline, preservatives, and the like. “Consistingof” shall mean excluding more than trace elements of other ingredientsand substantial method steps for administering the compositions of thisinvention. Embodiments defined by each of these transition terms arewithin the scope of this invention.

As used herein, the term “sodium deoxycholate” refers to sodium(4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoateas shown below. Other stereoisomers are within the scope of theinvention.

Sodium deoxycholate or sodium(4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoatecan be prepared according to the methods disclosed in U.S. PatentPublication No. 2008/0318870A1 entitled “Synthetic Bile AcidComposition, Method And Preparation” filed on Feb. 21, 2008, which ishereby incorporated by reference in its entirety.

As used herein, the term “aqueous pharmaceutical formulation” refers toa formulation of sodium deoxycholate in water suitable foradministration to a patient.

As used herein, the term “buffer” refers to an aqueous solutioncomprising a mixture of a weak acid and its conjugate base or a weakbase and its conjugate acid. A buffer has the property that the pH ofthe solution changes very little when a small amount of acid or base isadded to it. Buffer solutions are used as a means of keeping pH at anearly constant value in a wide variety of chemical applications.Examples of suitable buffers include phosphate buffers and those knownin the literature (see, for example, Troy, D. B., ed. (2005) Remington:The Science and Practice of Pharmacy, 21^(st) ed., Lippincott Williams &Wilkins).

As used herein, the term “base” refers to various typicallywater-soluble compounds, molecules or ions that in solution have a pHgreater than 7. Such compounds, molecules or ions are able to take up aproton from an acid or are able to give up an unshared pair of electronsto an acid. Examples of suitable bases include metal carbonates andbicarbonates, for example sodium carbonate, calcium carbonate, magnesiumcarbonate, zinc carbonate, sodium bicarbonate and the like; and metalhydroxides, for example sodium hydroxide, potassium hydroxide, and thelike, such as those known in the literature (see, for example, Troy, D.B., ed. (2005) Remington: The Science and Practice of Pharmacy, 21sted., Lippincott Williams & Wilkins).

As used herein, the term “metal carbonates” refers to the metal salt ofCO₃ ²⁻. For example, sodium carbonate, calcium carbonate, magnesiumcarbonate, zinc carbonate, and the like.

As used herein, the term “metal bicarbonates” refers to the metal saltof HCO₃ ⁻. For example, sodium bicarbonate, and the like.

As used herein, the term “metal hydroxides” refers to the metal salt of⁻OH. For example, sodium hydroxide, potassium hydroxide, and the like.

As used herein, the terms “sterile water” or “water for injection” referto a sterile, nonpyrogenic preparation of water for injection whichcontains no bacteriostat, antimicrobial agent or added buffer. Ingeneral, the osmolar concentration of additives totals at least 112mOsmol/liter (two-fifths of the normal osmolarity of the extracellularfluid −280 mOsmol/liter).

As used herein, the term “benzyl alcohol” refers to the compound

As used herein, the term “precipitation” refers to the formation of asolid in a solution. As used herein, the term “solution” refers to asubstantially homogeneous mixture comprising two or more substancesdissolved in a solvent.

As used herein, the term “substantially inhibit precipitation” means toinhibit most or all visible precipitation or maintain homogeneity. Thismay be done over a desired period of time.

As used herein, the term “relative standard deviation for homogeneity”or “H_(E)” refers to the value obtained by dividing the standarddeviation of the homogeneity by the absolute value of the mean. An H_(E)less than 10 indicates very good homogeneity.

Formulations

Knowledge about the chemical and physical stability of a drugformulation in the desired media for delivery is valuable. In the longerterm, the stability of the formulation will dictate the shelf life ofthe marketed product. It is preferable that the active ingredient in apharmaceutical formulation be at the required concentration whenadministered to a patient.

Current methods for the administration of sodium deoxycholate to apatient include the administration of a low concentration (i.e., <5%w/v) of an aqueous solution of sodium deoxycholate, as it has been shownthat the low concentration is beneficial for the effective and saferemoval of fat deposits in the body. However, it has been observed thata precipitate forms at both relatively low (i.e., <5% w/v) and high(i.e., >16% w/v) concentrations of sodium deoxycholate in aqueous media.This precipitation results in a limited shelf life of aqueous solutionsof sodium deoxycholate, even at cold temperatures (3-5° C.). Thisinstability of aqueous solutions of sodium deoxycholate can becircumvented by the preparation of an aqueous solution of sodiumdeoxycholate at a concentration of about 5% to about 16% w/v, and havingthe practitioner dilute the pharmaceutical composition of sodiumdeoxycholate just prior to use. Whereas this dilution method iseffective to allow for both storage stability and effective patientdosing, it is not ideal as a method for routine use.

It has been found that aqueous solutions of sodium deoxycholate at low(i.e., <5% w/v) concentrations of sodium deoxycholate can be stabilizedadjusting the pH of the solution. The present application is directed toan aqueous pharmaceutical formulation comprising less than about 5% w/vsodium deoxycholate wherein the formulation is maintained at a pHsufficient to substantially inhibit precipitation of the sodiumdeoxycholate. In some embodiments, the pharmaceutical formulation,comprising less than about 5% w/v sodium deoxycholate in water, oralternatively, less than about 4.5%, or alternatively, less than about4%, or alternatively, less than about 3.5%, or alternatively, less thanabout 3%, or alternatively, less than about 2.5%, or alternatively, lessthan about 2%, or alternatively, less than about 1.5%, or alternatively,less than about 1%, or alternatively, less than about 0.75%, oralternatively, less than about 0.5%, or alternatively, less than about0.1% w/v sodium deoxycholate in water.

Sodium deoxycholate or sodium(4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoatecan be prepared according to the methods disclosed in U.S. PatentPublication No. 2008/0318870A1 entitled “Synthetic Bile AcidComposition, Method And Preparation” filed on Feb. 21, 2008, which ishereby incorporated by reference in its entirety.

In one embodiment, the pharmaceutical formulations disclosed herein aresuitable for injection into a human. The method of injection can be anytype of injection, such as subcutaneous injection, as well as otherforms of injection. Therefore, in some embodiments, the aqueousformulation is sterile. The aqueous formulation can be prepared usingsterile water or water for injection (WFI).

In one aspect of the present invention, the precipitation of sodiumdeoxycholate is substantially inhibited for a period of at least aboutsix months. In another aspect, the precipitation of sodium deoxycholateis substantially inhibited for a period of at least about one year. Inyet another aspect, the precipitation of sodium deoxycholate issubstantially inhibited for a period of at least about two years.

It is contemplated that when stored at various temperatures, for exampleat ambient or cold temperatures, the formulation can have an increasedshelf life. In certain embodiments, the formulation is stored at atemperature of from about 17° C. to about 27° C. In some embodiments,the temperature of the formulation is increased to a temperature ofabout 25° C. to about 37° C. In other embodiments, the formulation isstored at a temperature of from about 2 ° C. to about 8° C.

In certain embodiments, the pH of the formulation ranges from about 8.0to about 8.5. In one embodiment, the pH of the formulation is about 8.0,or alternatively, about 8.1, or alternatively, about 8.2, oralternatively, about 8.3, or alternatively, about 8.4, or alternatively,about 8.5.

In one embodiment, the pH is established by the use of a base. It iscontemplated that any base can be used to increase the pH of theformulation provided that it does not react with the sodium deoxycholateand will not cause harm to the patient. In some embodiments, the base isselected from the group consisting of metal carbonates, metalbicarbonates, metal hydroxides, or a mixture thereof. Examples of basesinclude, but are not limited to, a base selected from the groupconsisting of sodium carbonate, calcium carbonate, magnesium carbonate,zinc carbonate, sodium bicarbonate, sodium hydroxide and potassiumhydroxide or a mixture thereof. In one embodiment, the base is sodiumhydroxide.

In certain cases, the pH of the formulation may be maintained with theuse of a buffer. Various buffers are known in the art and it iscontemplated that any buffer having buffering capacity at the desired pHcan be used in the formulations disclosed herein. In one embodiment, thebuffer is a phosphate buffer. The amount of phosphate in the formulationcan be determined to provide a desired pH and salt concentration. In oneembodiment, the formulation comprises about 10 mM phosphate.

In some embodiments, the formulation comprises at least one excipient toaid in achieving a formulation with desired properties, such asincreased solubility, preservability or to provide an isotonic solution.Such excipients are known in the art. In one embodiment, the formulationcomprises about 1% sodium chloride. In another embodiment, theformulation comprises about 1% benzyl alcohol. In some embodiments, theformulation comprises about 1% benzyl alcohol and about 1% sodiumchloride. In some embodiments, the formulation comprises about 0.9%benzyl alcohol and about 0.9% sodium chloride.

The formulations disclosed herein comprise less than about 5% w/v sodiumdeoxycholate in water maintained at a pH sufficient to substantiallyinhibit precipitation of the sodium deoxycholate. The amount ofprecipitation or homogeneity of the formulation can be measured usingvarious methods. For example, it can be measured quantitatively usinglight scattering by illuminating the formulation with aspectrophotometer. Or alternatively, the homogeneity can be measuredqualitatively by observing the visual clarity of the solution with theeye. In some embodiments, the formulation has a relative standarddeviation for homogeneity of less than about 5%. Alternatively, theformulation has a relative standard deviation for homogeneity of lessthan about 4%, or alternatively, about 3%, or alternatively, about 2%,or alternatively, about 1%.

The use of prodrugs of sodium deoxycholate in the formulations disclosedherein are also contemplated. A prodrug is an active or inactivecompound that is modified chemically through in vivo physiologicalaction, such as hydrolysis, metabolism and the like, into a compound ofthe embodiments following administration of the prodrug to a patient.For example, one may prepare an ester of the present deoxycholic acid ateither or both of the hydroxyl groups thereon or of suitable derivativesthereof, so that the release of the deoxycholic acid or derivativesthereof is triggered by the disruption of the cell membrane, and releaseof esterase. With the release of esterase, the ester protecting group iscleaved so that the deoxycholic acid active form or derivatives thereofis present at the desired location in situ. For a general discussion ofprodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).

In some embodiments, the solutions herein do not include lipids,phospholipids, or phosphatidylcholine. In some embodiments, thesolutions herein include up to 5% w/w, w/v, or v/v lipids, specificallyphospholipids, or more specifically phosphatidylcholine.

In some embodiments, the aqueous pharmaceutical formulation of theinvention can further comprise a second therapeutic agent selected fromthe group consisting of: anti-microbial agents, vasoconstrictors,anti-thrombotic agents, anti-coagulation agents, suds-depressants,anti-inflammatory agents, analgesics, dispersion agents, anti-dispersionagents, penetration enhancers, steroids, tranquilizers, musclerelaxants, and anti-diarrhea agents. In some embodiments, a solution isin a container that contains up to 500 mL of solution. Such containercan be a syringe or syringe-loadable container.

In some embodiments, the formulations further comprise a molecule knownto cause fat to die by an orthogonal mechanism. Such molecules includeneuropeptide Y (NPY) antagonists including, but not limited to, NPYreceptor antagonists, such as BIBP-3226 (Amgen), BIBO-3304 (BoehringerIngleheim), BMS-192548 and AR-H040922 (Bristol-Myers Squibb), LY-357897(Eli Lilly), 1229U91 and GW4380145 (GlaxoSmithKline), JNJ-5207787(Johnson & Johnson), Lu-AA-44608 (Lundbeck), MK-0557 (Merck NPY),NGD-95-1 (Neurgogen), NLX-E201 (Neurologix), CGP-71683 (Novartis),PD-160170 (Pfizer), SR-120819A, BIIE0246, and S.A.0204 (Sanofi Aventis),S-2367 (Shiongli), dihydropyridine and dihydropyridine derivatives thatare NPY receptor antagonists, bicyclic compounds that are NPY receptorantagonists, carbazole NPY receptor antagonists, and tricyclic compoundsthat are NPY receptor antagonists (See, e.g., WO 2006/133160 and U.S.6,313,128). Also contemplated are fat selective pro-apoptotic peptidessuch as the CKGGRAKDC peptide that homes to white fat vasculature (See,Kolonin M. G. et al., Nat. Med., 2004, 10(6): 625-32).

Another aspect of the invention relates to mixing adipo-ablative bileacids, such as, deoxycholic acid (DCA) with agents that kill fat cells.In one aspect, this invention contemplates a means to enhance theaesthetic effects of deoxycholate injections by mixing into thedeoxycholate injectate a molecule that causes fat to die by anorthogonal mechanism. Examples of such candidate molecules include, butare not limited to, neuropeptide Y (NPY) antagonists and fat selectivepro-apoptotic peptides. Since both fat cell killing and skin tighteningmay be required to mediate the desired effects, the effects of an agentwith fat killing ability and potent skin tightening effects (such asdeoxycholate) can be enhanced via the addition of a molecule with potentfat cell killing effects. Additionally, molecules that require access tothe vasculature to kill (such as certain pro-apoptotic peptides thatbind to proteins expressed on the luminal side of capillaries) can gainaccess to these proteins because deoxycholate may cause vascularleakage. Thus, such agents can be synergistic with deoxycholatepotentially creating a more potent means to mediate body contouring infewer therapeutic sessions.

Examples of NPY antagonists include, but are not limited to, NPYreceptor antagonists, such as BIBP-3226 (Amgen), BIBO-3304 (BoehringerIngleheim), BMS-192548 and AR-H040922 (Bristol-Myers Squibb), LY-357897(Eli Lilly), 1229U91 and GW4380145 (GlaxoSmithKline), JNJ-5207787(Johnson & Johnson), Lu-AA-44608 (Lundbeck), MK-0557 (Merck NPY),NGD-95-1 (Neurgogen), NLX-E201 (Neurologix), CGP-71683 (Novartis),PD-160170 (Pfizer), SR-120819A, BIIE0246, and S.A.0204 (Sanofi Aventis),S-2367 (Shiongli), dihydropyridine and dihydropyridine derivatives thatare NPY receptor antagonists, bicyclic compounds that are NPY receptorantagonists, carbazole NPY receptor antagonists, and tricyclic compoundsthat are NPY receptor antagonists. See, e.g., WO 2006/133160 and U.S.Pat. No. 6,313,128 (incorporated herein by reference in its entiretyincluding figures).

Exemplary fat selective pro-apoptotic peptides includes, but is notlimited to, CKGGRAKDC peptide that homes to white fat vasculature. See,Kolonin M. G. et al., Nat. Med. June 10(6):625-32 (2004).

Sodium deoxycholate or sodium(4R)-4-((3R,5R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoatecan be prepared according to the methods disclosed in U.S. PatentPublication No. 2008/0318870A1 entitled “Synthetic Bile AcidComposition, Method And Preparation” filed on Feb. 21, 2008, which ishereby incorporated by reference in its entirety. It will be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Methods

Disclosed herein is a method for inhibiting precipitation of sodiumdeoxycholate in an aqueous solution comprising less than about 5% w/v ofsodium deoxycholate, said method comprising maintaining pH of thesolution from at least about 8.0 to about 8.5.

In one aspect of the present invention, methods disclosed hereinsubstantially inhibit the precipitation of sodium deoxycholate insolution over a period of at least about six months. In another aspect,the precipitation of sodium deoxycholate is substantially inhibited fora period of at least about one year. In yet another aspect, theprecipitation of sodium deoxycholate is substantially inhibited for aperiod of at least about two years.

It has been found that the pH of the solution can inhibit theprecipitation of sodium deoxycholate at concentrations of less thanabout 5% w/v in water allow the sodium deoxycholate to be maintained insolution. In one embodiment, the pH is established by the use of a base.It is contemplated that any base can be used to increase the pH of theformulation provided that it does not react with the sodiumdeoxycholate. In some embodiments, the base is selected from the groupconsisting of metal carbonates, metal bicarbonates, and metalhydroxides, or a mixture thereof. Examples of bases include, but are notlimited to, a base selected from the group consisting of sodiumcarbonate, calcium carbonate, magnesium carbonate, zinc carbonate,sodium bicarbonate, sodium hydroxide and potassium hydroxide or amixture thereof. In one embodiment, the base is sodium hydroxide.

In certain embodiments, the pH ranges from about 8.0 to about 8.5. Inone embodiment, the pH of the formulation is about 8.0, oralternatively, about 8.1, or alternatively, about 8.2, or alternatively,about 8.3, or alternatively, about 8.4, or alternatively, about 8.5. Inone embodiment, the pH of the aqueous solution is about 8.3.

In certain cases, the pH of the formulation may need to be maintainedwith the use of a buffer. Various buffers are know in the art and it iscontemplated that any buffer having buffering capacity at the desired pHcan be used in the formulations disclosed herein. In one embodiment, thebuffer is a phosphate buffer. The amount of phosphate required toprovide a desired pH and salt concentration can be calculated usingmethods well known in the art. In one embodiment, the formulationcomprises about 10 mM phosphate.

In one embodiment, the methods disclosed herein provide formulationswhich are suitable for injection into a human. The method of injectioncan be any type of injection, such as subcutaneous injection, as well asother forms of injection. Therefore, in some embodiments, the aqueoussolution comprises sterile water or water for injection (WFI).

In one aspect, it may be that one or more excipients are used tomaintain the solubility, or increase the preservability of sodiumdeoxycholate present in the formulation. In one embodiment, the methodcomprises adding about 1% benzyl alcohol. In some embodiments, theformulation also comprises at least one excipient to aid in achieving anisotonic solution. Such excipients are known in the art. In oneembodiment, the method comprises adding about 1% sodium chloride. Insome embodiments, the method comprises adding both 1% benzyl alcohol and1% sodium chloride. In some embodiments, the method comprises addingboth 0.9% benzyl alcohol and 0.9% sodium chloride. Using the methodsdisclosed herein, an aqueous solution comprising less than about 5% w/vsodium deoxycholate is maintained at a pH sufficient to substantiallyinhibit precipitation of the sodium deoxycholate. The amount ofprecipitation or homogeneity of the formulation can be measured usingvarious methods. For example, it can be measured quantitatively bymeasuring the light scattering via illumination by a spectrophotometer.Or alternatively, the homogeneity can be measured qualitatively bysimply observing the visual clarity of the solution with the eye. Insome embodiments, the method provides a pharmaceutical formulationhaving a relative standard deviation for homogeneity of less than about5%. Alternatively, the relative standard deviation for homogeneity ofless than about 4%, or alternatively, about 3%, or alternatively, about2%, or alternatively, about 1%.

The storage temperature can assist in maintaining the solubility of thesodium deoxycholate of the formulation. In certain embodiments, thestorage temperature is from about 17° C. to about 27° C. In someembodiments, the storage temperature is about 25° C. to about 37° C. Inother embodiments, the storage temperature is from about 2° C. to about8° C.

It is contemplated that the sodium deoxycholate concentration can varyfrom about 0.05% w/v to about 5% w/v without effecting solubility in theformulation. In certain embodiments, the formulation comprises a sodiumdeoxycholate concentration of about 0.05% w/v. Alternatively, theformulation comprises a sodium deoxycholate concentration of about 0.07%w/v, or alternatively, about 0.1% w/v, or alternatively, about 0.3% w/v,or alternatively, about 0.5% w/v, or alternatively, about 0.7% w/v, oralternatively, about 1% w/v, or alternatively, about 2% w/v, oralternatively, about 3% w/v, or alternatively, about 4% w/v, oralternatively, about 5% w/v.

EXAMPLES

In the examples and elsewhere in the specification, abbreviations havethe following meanings:

-   -   A₅₀₀=Absorbance at 500 Nanometers    -   cc=Cubic centimeter    -   cm=Centimeter    -   F/T=Freeze/Thaw    -   HPLC=High-Performance Liquid Chromatography    -   hr.=Hour    -   mg=Milligram    -   mL=Milliliter    -   mm=Millimeter    -   mM=Millimolar    -   nm=Nanometer    -   t=Time    -   UV=Ultraviolet    -   v/v=Volume /Volume    -   w/v=Weight/Volume (g/mL)    -   w/w=Weight/Weight    -   WFI=Water for Injection

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Althoughseveral embodiments of the invention are described herein in detail, itwill be understood by those skilled in the art that variations may bemade thereto without departing from the spirit of the invention or thescope of the appended claims. Such modifications fall within the scopeof the appended claims.

Example 1 Changes in pH and Visual Observations

This example demonstrates that aqueous formulations containing less than5% w/v sodium deoxycholate forms a precipitate at various temperatureswhich cannot be brought back into solution. Solubility was best inaqueous formulations containing 5% w/v.

The stability of three lots of sodium deoxycholate dissolved in 0.9%benzyl alcohol and water for injection (WFI) was examined for changes inpH and visual observations, such as the formation of precipitate. Eachlot was formulated at four concentrations: 5, 50, 100 and 160 mg/mL(0.5, 5, 10 and 16% w/v, respectively) dissolved in 0.9% benzyl alcoholand WFI. Table 1 provides a summary of the variables that were tested inthis example.

TABLE 1 Experimental Variables Sample (Lot) Sodium Deoxycholate %Weight/Volume Number Concentration (mg/mL) (% w/v) 1A 5 0.5 1B 50 5 1C100 10 1D 160 16 2A 5 0.5 2B 50 5 2C 100 10 2D 160 16 3A 5 0.5 3B 50 53C 100 10 3D 160 16 4 0 0

The solutions were prepared as follows. Two samples for each conditionwere filled at 2.0 mL into sterile 3 c.c. glass vials (West Pharma P/N68000316), and stoppered with sterile 13 mm serum stoppers (West PharmaP/N 19560001). These samples were analyzed in duplicate at each timepoint in order to verify any changes in pH and visual observations,during storage at 2-8° C. and 25° C. for two weeks. Table 2 summarizesthe conditions and time points at which samples were analyzed throughoutthe study.

TABLE 2 Conditions and Time Points Time Point Storage ConditionsAnalyses Analytical Methods 2-8° C. 1, 7, 14 days pH and visualobservation  25° C. 0, 1, 7, 14 days pH and visual observation

As seen in Tables 3 and 4, at 5 mg/mL (0.5% w/v), fine particulates wereseen generally across all lots and at both 4° C. and 25° C., thoughSample 1 at 4° C. did not precipitate. At 50 mg/mL (5% w/v), all samplesand conditions were clear. At 100 mg/mL (10% w/v), there was atemperature effect as all lots at 4° C. precipitated out while the 25°C. lots remained clear. At 160 mg/mL (16% w/v), there was a temperatureeffect as all lots at 4° C. precipitated out while the 25° C. lotsremained clear. The precipitation observed at 160 mg/mL (16% w/v) wasmore severe and rapid than 100 mg/mL, however, the precipitated productat these two concentrations could be resolubilized after warming to roomtemperature and vortexing. The precipitated material at 5 mg/mL (0.5%w/v) could not be brought back into solution. This data is showngraphically in FIGS. 1A to 1F.

The pH profiles for both 4° C. and 25° C. were overlapping, so itappears that temperature did not have an impact at concentrations lessthan 50 mg/mL (5% w/v). Instead, there appears to be a solubility pH forformulations less than 50 mg/mL (5% w/v). At 100 and 160 mg/mL, the 4°C. samples had a higher pH than the 25° C. samples. However, withincreasing concentration, temperature appears to become more importantthan pH, and the formulation is increasingly sensitive to temperature.

TABLE 3 pH over Time at 4° C. (Average of Two Observations) SampleSodium % (Lot) Deoxycholate Weight/Volume Number Concentration (% w/v) 0days 1 day 7 days 14 days 1A  5 mg/mL 0.5 7.565 7.56 7.625 7.67 1B  50mg/mL 5 7.885 7.905 7.95 7.965 1C 100 mg/mL 10 7.89 7.93 7.995 8Precipitate Precipitate 1D 160 mg/mL 16 7.865 8.14 8.045 8.055Precipitate Precipitate Precipitate 2A  5 mg/mL 0.5 7.62 7.53 7.6 7.66Precipitate Precipitate Precipitate 2B  50 mg/mL 5 7.775 7.835 7.8657.88 2C 100 mg/mL 10 7.77 7.845 7.98 7.93 Precipitate PrecipitatePrecipitate 2D 160 mg/mL 16 7.745 8 8.15 7.975 Precipitate PrecipitatePrecipitate 3A  5 mg/mL 0.5 7.625 7.61 7.715 7.74 PrecipitatePrecipitate Precipitate 3B  50 mg/mL 5 7.91 7.945 7.99 8 3C 100 mg/mL 107.905 7.97 7.99 8.01 Precipitate Precipitate 3D 160 mg/mL 16 7.86 8.1158.15 8.065 Precipitate Precipitate Precipitate 4  0 Placebo 0 6.945 7.087.13 6.87

TABLE 4 pH over Time at 25° C. (Average of Two Observations) SampleSodium % (Lot) Deoxycholate Weight/Volume Number Concentration (% w/v) 0days 1 day 7 days 14 days 1A  5 mg/mL 0.5 7.565 7.595 7.7 7.73Precipitate Precipitate Precipitate 1B  50 mg/mL 5 7.885 7.95 7.95 7.981C 100 mg/mL 10 7.89 7.955 7.945 7.975 1D 160 mg/mL 16 7.865 7.94 7.9357.93 2A  5 mg/mL 0.5 7.62 7.56 7.665 7.69 Precipitate PrecipitatePrecipitate 2B  50 mg/mL 5 7.775 7.845 7.865 7.885 2C 100 mg/mL 10 7.777.85 7.865 7.875 2D 160 mg/mL 16 7.745 7.815 7.865 7.83 3A  5 mg/mL 0.57.625 7.63 7.71 7.76 Precipitate Precipitate Precipitate 3B  50 mg/mL 57.91 7.955 7.985 8.015 3C 100 mg/mL 10 7.905 7.965 7.98 8.005 3D 160mg/mL 16 7.86 7.965 7.955 7.975 4 0 Placebo 0 6.945 6.825 6.945 6.93

Table 5 shows that the solubility of sodium deoxycholate is independentof the lot to lot variability in pH. In general, there was no differencein physical stability across lots under like conditions.

TABLE 5 pH by Sample over Time Sample (Lot) Sodium % Number-Deoxycholate Weight/Volume Temperature Concentration (% w/v) 0 days 1day 7 days 14 days  1A-4° C.  5 mg/mL 0.5 7.565 7.56 7.625 7.67  2A-4°C.  5 mg/mL 0.5 7.62 7.53 7.6 7.66  3A-4° C.  5 mg/mL 0.5 7.625 7.617.715 7.74  1B-4° C.  50 mg/mL 5 7.885 7.905 7.95 7.965  2B-4° C.  50mg/mL 5 7.775 7.835 7.865 7.88  3B-4° C.  50 mg/mL 5 7.91 7.945 7.99 8 1C-4° C. 100 mg/mL 10 7.89 7.93 7.995 8  2C-4° C. 100 mg/mL 10 7.777.845 7.98 7.93  3C-4° C. 100 mg/mL 10 7.905 7.97 7.99 8.01  1D-4° C.160 mg/mL 16 7.865 8.14 8.045 8.055  2D-4° C. 160 mg/mL 16 7.745 8 8.157.975  3D-4° C. 160 mg/mL 16 7.86 8.115 8.15 8.065 1A-25° C.  5 mg/mL0.5 7.565 7.595 7.7 7.73 2A-25° C.  5 mg/mL 0.5 7.62 7.56 7.665 7.693A-25° C.  5 mg/mL 0.5 7.625 7.63 7.71 7.76 1B-25° C.  50 mg/mL 5 7.8857.95 7.95 7.98 2B-25° C.  50 mg/mL 5 7.775 7.845 7.865 7.885 3B-25° C. 50 mg/mL 5 7.91 7.955 7.985 8.015 1C-25° C. 100 mg/mL 10 7.89 7.9557.945 7.975 2C-25° C. 100 mg/mL 10 7.77 7.85 7.865 7.875 3C-25° C. 100mg/mL 10 7.905 7.965 7.98 8.005 1D-25° C. 160 mg/mL 16 7.865 7.94 7.9357.93 2D-25° C. 160 mg/mL 16 7.745 7.815 7.865 7.83 3D-25° C. 160 mg/mL16 7.86 7.965 7.955 7.975

Three factors have been identified to contribute to solubility. In noparticular order these are concentration, pH and temperature. It hasbeen found that the lot to lot variability in pH does not account forbetter or poorer solubility. Overall, the 50 mg/mL seems to be the mostrobust condition, where all lots appear to be above the “solubility” pH,and where temperature has no impact within the two-week time frame ofthis study. The results suggest that concentration, pH, and storagetemperature factor into the stability of sodium deoxycholate. Thesamples at 50 mg/mL showed the best stability at 4° C. and 25° C. fortwo weeks.

Example 2 Stability Study

This example demonstrates that aqueous formulations of less than 5% w/vsodium deoxycholate at a pH of 8.0 to 8.5 exhibited very goodsolubility. This example also demonstrates that sodium deoxycholateshows no degradation in aqueous formulations having less than 5% w/vsodium deoxycholate during storage for eight weeks at 4° C., 25° C. and37° C. A number of formulations were subjected to agitation, UVexposure, and freeze-thaw cycles and exhibited no degradation orprecipitation when exposed to those stresses.

A two-month stability study of sodium deoxycholate in several differentformulations was conducted. By reverse phase HPLC, sodium deoxycholateshowed no degradation in all formulations at all temperatures. However,solubility was best in formulations containing 10 mM phosphate, 0.9%NaCl, 0.9% Benzyl Alcohol at a pH of 8.0 or 8.5. Storage temperatures of25° C. and 37° C. also assisted solubility. All formulations that werechosen for agitation, UV exposure, and freeze-thaw exhibited nodegradation or precipitation when exposed to those stresses.

Table 6 provides a list of chemicals used in the preparation of theformulations disclosed herein.

TABLE 6 Chemicals used in formulation preparation Manufacturer Chemical(Product Number) Grade Benzyl Alcohol J. T. Baker (9041-01) N.F./MultiCompendial Sodium Chloride EMD Chemicals (7760) Molecular Biology Grade(99.95% pure) Sodium Hydroxide J. T. Baker (3728-01) N.F./F.C.C. PelletsSodium Phosphate J. T. Baker (3820-01) U.S.P./F.C.C. Monobasic,Monohydrate Sorbitol (Extra Pure) EM Science (0035835B) N.F./Ph. Eur.

Stability Studies

The stability of the sodium deoxycholate formulations was examined atthe conditions summarized in Table 7. Reverse phase HPLC was used alongwith visual observations which were conducted at each time point tomonitor any precipitation or cloudiness. The formulations tested forvortex, UV exposure, and freeze-thaw, were chosen from the formulationsthat performed the best in the temperature study. The major stabilityissues of sodium deoxycholate observed during this study were solubilityand pH drift.

TABLE 7 Stresses and time points Stress Conditions Time PointsTemperature  4° C. 0, 2 weeks, 1 and 2 months 25° C. 0, 2 weeks, 1 and 2months 37° C. 0, 2 weeks, 1 and 2 months Agitation Vortex 4 hours LightUV exposure 24 hours Freeze-Thaw −70° C. to 25° C. 5 cycles

i. Effect of Temperature on Stability

The formulations were incubated at 4° C., 25° C. and 37° C. and analyzedfor a) degradation products, b) pH stability and c) formulation clarity.

Formulations Tested

Except for formulations only consisting of water, sodium deoxycholatewas dissolved in buffer that had been titrated to a pH of 6. Eachformulation, except for the two only consisting of water, was thentitrated to its target pH with NaOH. All formulations were sterilefiltered and filled to 1.5 mL in sterile 2 cc glass vials. The filledvials were then autoclaved. The formulations 3A and 3B were cloudy priorto filling.

TABLE 8 List of Formulations Sodium Deoxycholate % Weight/ ConcentrationVolume Formulation Buffer Excipient(s) pH (mg/mL) (% w/v) 1A None WFIonly — 20 2 WFI20 1B None WFI only — 5 0.5 WFI5 2A 10 mM 0.9% NaCl; 7.520 2 P7.5NB20 phosphate 0.9% Benzyl Alcohol 2B 10 mM 0.9% NaCl; 7.5 50.5 P7.5NB5 phosphate 0.9% Benzyl Alcohol 3A 10 mM 5% Sorbitol; 7.5 20 2P7.5SB20 phosphate 0.9% Benzyl Alcohol 3B 10 mM 5% Sorbitol; 7.5 5 0.5P7.5SB5 phosphate 0.9% Benzyl Alcohol 4A 10 mM 0.9% NaCl; 8.0 20 2P8NB20 phosphate 0.9% Benzyl Alcohol 4B 10 mM 0.9% NaCl; 8.0 5 0.5 P8NB5phosphate 0.9% Benzyl Alcohol 4C 10 mM 0.9% NaCl; 8.0 10 1 P8NB10phosphate 0.9% Benzyl Alcohol 5 10 mM 0.9% NaCl 8.0 5 0.5 P8N5 phosphate6A 10 mM 5% Sorbitol; 8.0 20 2 P8SB20 phosphate 0.9% Benzyl Alcohol 6B10 mM 5% Sorbitol; 8.0 5 0.5 P8SB5 phosphate 0.9% Benzyl Alcohol 7 10 mM5% Sorbitol 8.0 5 0.5 P8S5 phosphate 8A 10 mM 0.9% NaCl; 8.5 20 2P8.5NB20 phosphate 0.9% Benzyl Alcohol 8B 10 mM 0.9% NaCl; 8.5 5 0.5P8.5NB5 phosphate 0.9% Benzyl Alcohol 9A 10 mM 5% Sorbitol; 8.5 20 2P8.5SB20 phosphate 0.9% Benzyl Alcohol 9B 10 mM 5% Sorbitol; 8.5 5 0.5P8.5SB5 phosphate 0.9% Benzyl Alcohola) Degradation Products

Reverse phase HPLC was used to visualize degradation products. A samplechromatogram of formulation 4A shows the main peak with respect to thedegradation products (FIG. 3). FIG. 4 shows the main peak with respectto the degradation products (i.e. Purity) for all formulations at theincubation temperatures of 4° C., 25° C. and 37° C. FIG. 4 shows thepurity at t=0 and 2 months. The results suggest that no chemicaldegradation occurred in any of the formulations of Table 8.

b) pH Stability

Table 9 shows the pH of the formulations prior to filling and at t=0. Ingeneral, the pH drifted towards 8 and remained constant throughout thestudy. This suggests that sodium deoxycholate has some bufferingcapacity.

TABLE 9 pH Drift Formulation pH prior to fill pH at t = 0 WFI20 8.1 8.3WFI5 7.2 7.5 P7.5NB20 7.5 7.7 P7.5NB5 7.5 7.8 P7.5SB20 7.5 7.6 P7.5SB57.4 7.6 P8NB20 8.0 8.3 P8NB5 8.0 8.3 P8N5 8.1 8.5 P8NB10 8.0 8.2 P8SB207.9 8.0 P8SB5 8.0 7.8 P8S5 8.0 7.9 P8.5NB20 8.5 8.3 P8.5NB5 8.5 8.5P8.5SB20 8.5 8.1 P8.5SB5 8.5 8.0c) Formulation Clarity

The clarity of each formulation was determined by two methods. Onemethod was by visual observation, where the vial was held up to anintense light and the presence of particles was determined by what theeye could see. A set of vials filled with the formulation buffers andincubated at the three temperatures where used as reference standards.The other method used light scattering by illuminating the samplesacross a 1 cm path length with 500 nm light in a spectrophotometer.

The presence of visible precipitates in some formulations increased overtime, particularly at 4° C. The formulations containing sorbitol at thelowest pH produced the most precipitation. The formulations thatperformed the best contained 10 mM phosphate, 0.9% NaCl, 0.9% BenzylAlcohol at pH 8.0 or 8.5.

Tables 10, 11, 12 and 13 show the clarity results. The “Visual Clarity”column has a ranking system as follows: 0=clear, no particles; 1=clear,few particles; 2=clear, several particles; 3=clear, many largeparticles, 4=slightly cloudy; 5=very cloudy.

TABLE 10 t = 0 Formulation Visual Clarity Comments A₅₀₀ WFI20 0 0.0064WFI5 0 0.0103 P7.5NB20 0 (gelatinous) 0.0467 P7.5NB5 0 0.0042 P7.5SB20 0(gelatinous) 0.0405 P7.5SB5 3 Many large 0.1599 particles P8NB20 00.0076 P8NB5 0 0.0059 P8N5 0 0.0053 P8NB10 0 0.0038 P8SB20 0 0.0067P8SB5 0 0.0426 P8S5 0 0.0099 P8.5NB20 0 0.0012 P8.5NB5 0 0.0048 P8.5SB200 0.0130 P8.5SB5 0 0.0230

TABLE 11 4° C., 2 months Visual Formulation Clarity Comments A₅₀₀ WFI200 −0.0006 WFI5 2 Precipitate 0.0037 P7.5NB20 0 Gelatinous −0.0001P7.5NB5 0 −0.0036 P7.5SB20 3 Precipitate 0.0340 P7.5SB5 3 Precipitate0.2009 P8NB20 2 Possible −0.0006 precipitate P8NB5 0 −0.0019 P8N5 1Similar to buffer 0.0019 P8NB10 0 0.0124 P8SB20 2 Possible 0.0151precipitate P8SB5 2 Precipitate 0.0207 P8S5 0 0.0156 P8.5NB20 0 0.0147P8.5NB5 1 Similar to buffer 0.0156 P8.5SB20 1 Similar to buffer 0.0190P8.5SB5 0 0.0223

TABLE 12 25° C., 2 months Visual Formulation Clarity Comments A₅₀₀ WFI200 0.0078 WFI5 2 Precipitate 0.0102 P7.5NB20 0 0.0081 P7.5NB5 0 0.0161P7.5SB20 4 Precipitate 0.2129 P7.5SB5 4 Precipitate 0.1659 P8NB20 00.0179 P8NB5 1 Similar to buffer 0.0200 P8N5 0 0.0189 P8NB10 1 Similarto buffer 0.0161 P8SB20 0 0.0183 P8SB5 2 Possible precipitate 0.0262P8S5 1 Similar to buffer 0.0184 P8.5NB20 0 0.0208 P8.5NB5 1 Similar tobuffer 0.0195 P8.5SB20 0 0.0254 P8.5SB5 1 Similar to buffer 0.0229

TABLE 13 37° C., 2 months Visual Formulation Clarity Comments A₅₀₀ WFI200 0.0187 WFI5 2 Precipitate 0.0266 P7.5NB20 0 0.0196 P7.5NB5 0 0.0225P7.5SB20 4 Precipitate 0.4785 P7.5SB5 4 Precipitate 0.6743 P8NB20 00.0046 P8NB5 0 −0.0016 P8N5 1 Similar to buffer 0.0047 P8NB10 0 −0.0012P8SB20 0 0.0037 P8SB5 2 Possible 0.0195 precipitate P8S5 0 0.0086P8.5NB20 0 0.0013 P8.5NB5 0 0.0039 P8.5SB20 0 0.0056 P8.5SB5 1 Similarto buffer 0.0040

ii. Effects of Agitation, UV Exposure, and Freeze-Thaw on Stability

Based on the results from the temperature study, the followingformulations in Table 14 were chosen to test the effects of Agitation,UV Exposure, and Freeze-Thaw on stability.

TABLE 14 Agitation, UV Exposure, and Freeze - Thaw Formulations %Weight/ Concentration Volume Formulation Buffer Excipients pH (mg/mL) (%w/v) 4A 10 mM 0.9% NaCl; 8.0 20 2 P8NB20 phosphate 0.9% Benzyl Alcohol4C 10 mM 0.9% NaCl; 8.0 10 1 P8NB10 phosphate 0.9% Benzyl Alcohol 4B 10mM 0.9% NaCl; 8.0 5 0.5 P8NB5 phosphate 0.9% Benzyl Alcohol 8A 10 mM0.9% NaCl; 8.5 20 2 P8.5NB20 phosphate 0.9% Benzyl Alcohol 8C 10 mM 0.9%NaCl; 8.5 10 1 P8.5NB10 phosphate 0.9% Benzyl Alcohol 8B 10 mM 0.9%NaCl; 8.5 5 0.5 P8.5NB5 phosphate 0.9% Benzyl Alcohola) Degradation Products

Reverse phase HPLC was used to visualize degradation products. FIGS. 5,6 and 7 show the main peak with respect to the degradation products(i.e. Purity) for the formulations in Table 14 under the stress ofagitation, UV exposure, and five freeze-thaw cycles. The results suggestthat no chemical degradation occurred as a result of any of thestresses.

b) pH Stability

Table 15 shows the pH of the formulations prior to filling differentthan the pH's after opening the vials.

TABLE 15 pH Stability Formulation pH Prior to Fill pH at t = 0 4A 8.07.8 P8NB20 4C 8.0 7.9 P8NB10 4B 8.0 7.9 P8NB5 8A 8.5 8.3 P8.5NB20 8C 8.58.2 P8.5NB10 8B 8.5 8.1 P8.5NB5c) Formulation Clarity

The clarity of each formulation was determined by two methods. Onemethod was by visual observation, where the vial was held up to anintense light and the presence of particles was determined by what theeye could see. A set of vials filled with the formulation buffers andincubated at the three temperatures where used as reference standards.The other method used light scattering by illuminating the samplesacross a 1 cm path length with 500 nm light in a spectrophotometer.

Agitation, UV exposure, and freeze-thaw cycles does not appear to induceprecipitation.

Tables 16, 17 and 18 show the clarity results. The “Visual Clarity”column has a ranking system as follows: 0=clear, no particles; 1=clear,few particles; 2=clear, several particles; 3=clear, many largeparticles, 4=slightly cloudy; 5=very cloudy.

TABLE 16 Agitation Visual Formulation Stress Clarity Comments A₅₀₀ 4AControl 1 Similar to 0.0023 P8NB20 buffer P8NB20 4 hr. 0 0.0068 4CControl 0 −0.0001 P8NB10 P8NB10 4 hr. 0 0.0079 4B Control 0 0.0002 P8NB5P8NB5 4 hr. 0 0.0019 8A Control 0 −0.0002 P8.5NB20 P8.5NB20 4 hr. 00.0024 8C Control 1 Similar to 0.0087 P8.5NB10 buffer P8.5NB10 4 hr. 1Similar to 0.0010 buffer 8B Control 1 Similar to 0.0029 P8.5NB5 bufferP8.5NB5 4 hr. 0 0.0022

TABLE 17 UV Exposure Visual Formulation Stress Clarity Comments A₅₀₀ 4AControl 0 0.0039 P8NB20 P8NB20 24 hr. 0 0.0023 4C Control 1 Similar to0.0009 P8NB10 buffer P8NB10 24 hr. 1 Similar to 0.0011 buffer 4B Control0 0.0020 P8NB5 P8NB5 24 hr. 1 Similar to 0.0017 buffer 8A Control 00.0046 P8.5NB20 P8.5NB20 24 hr. 0 0.0072 8C Control 1 Similar to 0.0121P8.5NB10 buffer P8.5NB10 24 hr. 0 0.0023 8B Control 1 Similar to 0.0027P8.5NB5 buffer P8.5NB5 24 hr. 0 0.0029

TABLE 18 Freeze - Thaw Cycles Formulation Stress Visual Clarity CommentsA₅₀₀ 4A t = 0 1 Similar to buffer 0.0023 P8NB20 P8NB20 F/T 5 0 0.0084 4Ct = 0 0 −0.0001 P8NB10 P8NB10 F/T 5 0 0.0064 4B t = 0 0 0.0002 P8NB5P8NB5 F/T 5 0 0.0022 8A t = 0 0 −0.0002 P8.5NB20 P8.5NB20 F/T 5 0 0.01618C t = 0 1 Similar to buffer 0.0087 P8.5NB10 P8.5NB10 F/T 5 0 −0.0005 8Bt = 0 1 Similar to buffer 0.0029 P8.5NB5 P8.5NB5 F/T 5 0 −0.0036

After screening the stability profile of sodium deoxycholate inseventeen unique formulations, it was determined that formulationscontaining 10 mM Phosphate, 0.9% NaCl and 0.9% Benzyl Alcohol at pH 8.0or 8.5 best prevented visible precipitation in addition to maximizingsolubility. By reverse phase HPLC, sodium deoxycholate shows nodegradation in all formulations at all temperatures, during storage foreight weeks at 4° C., 25° C. and 37° C. As far as visual clarity offormulations, storage temperatures of 25° C. and 37° C. actuallyimproved the solubility of sodium deoxycholate as compared to 4° C.

All formulations that were chosen for agitation, UV exposure, andfreeze-thaw exhibited no degradation or precipitation when exposed tothose stresses. Upon completion of the eight week storage stabilitystudy, selected formulations were subjected to these acute stressconditions. These experiments help determine the stability of sodiumdeoxycholate during the stress of common manufacturing and shippingprocedures. The data obtained from these acute stress studies confirmthe stability of sodium deoxycholate in formulations containing 10 mMPhosphate, 0.9% NaCl and 0.9% Benzyl Alcohol at pH 8.0 or 8.5, withsodium deoxycholate concentration of 5, 10 and 20 mg/mL.

1. An aqueous pharmaceutical formulation consisting of from about 0.5%w/v to about 1.5% w/v deoxycholate and at least one pharmaceuticallyacceptable excipient and/or carrier wherein the formulation has a pH ofabout 8.3.
 2. The aqueous pharmaceutical formulation of claim 1, whereinthe aqueous formulation is suitable for subcutaneous injection.
 3. Theaqueous pharmaceutical formulation of claim 1, wherein said at least onepharmaceutically acceptable excipient and/or carrier is selected fromthe group consisting of water, a buffer, and a preservative.
 4. Theaqueous pharmaceutical formulation of claim 2, wherein said at least onepharmaceutically acceptable excipient and/or carrier is selected fromthe group consisting of water, a buffer, and a preservative.